(1) Field of the Invention
The present application generally relates to fluorescent dyes. More specifically, the invention is directed to novel dyes useful for labeling nucleic acids and other molecules. These dyes contain a pentavalent phosphorus or arsenic atom in the ring system.
(2) Description of the Related Art
Numerous fluorescent dyes are available that are useful for labeling nucleic acids, proteins and other molecules. See, e.g., U.S. Pat. Nos. 6,184,379 and 6,552,199; European Patent Publications 0 543 333 and 0 567 622, and references cited therein.
There are a variety of properties that might be desirable for dyes that are intended for use as markers for detection of proteins or nucleic acids. These include the ability to bind to a protein, lipid or nucleic acid, the capability of incorporation into nucleic acids by enzymatic means when attached to a nucleotide, a lack of steric hindrance that could potentially interfere with nucleic acid hybridization, water solubility, lack of aggregation, ability to intercalate into double-stranded nucleic acids, and the presence of a reactive group that allows attachment of the dye to a nucleotide or other desirable target. Suitable dyes could have many of these properties but do not need to have them all. For instance, the ability to intercalate may allow detection of hybridization events in the presence of unhybridized probes or it may provide increased hybridization stabilization. Examples of these applications are disclosed in European Patent Publication EP 0 231 495 and U.S. Pat. Nos. 5,994,056 and 6,174,670. Similarly, the ability of a nucleotide labeled with the dye to be incorporated into a nucleic acid by an enzyme is a useful property when carrying out enzymatic labeling of nucleic acids. However, labels that are inhibitory towards incorporation can still be used in some methods where nucleic acids are chemically synthesized rather than using enzymatic means. Also, nucleotides with reactive groups such as allyl-amine may be incorporated enzymatically into nucleic acids and then in a second step they are post-synthetically modified by attachment of dyes. Steric hindrance may be compensated to some degree by utilizing a properly designed linker joining the dye to a nucleotide. For a discussion of this last point, see U.S. Patent Publication 2003/0225247.
The particular spectral characteristics of dyes are also important qualities. Although broad-spectrum white light can be used as a source of excitation, lasers with defined set wavelengths are most commonly employed. As such, dyes having most immediate use generally have excitation wavelengths at or near that of such standard laser wavelengths. Emission wavelengths are more flexible since filters can be used to isolate a particular part of the spectrum.
There are a number of machines available for detection of labeled nucleic acids that have been designed with dyes that are commonly used. For instance, there are several slide scanners that have been optimized for detection of nucleic acids labeled with the Cy3 and Cy5 dyes, as described in U.S. Pat. No. 5,268,486. However, the availability of dyes that have useful properties but have wavelengths that are not commonly used can be utilized by adopting lasers with compatible wavelengths.
A set of dyes with well separated emission spectra may be used simultaneously. Applications that utilize multiple dyes are immunostaining for various proteins in cells, in situ hybridization for multiple targets, non-radioactive sequencing, nucleic acid array analysis, protein array analysis, and non-specific cellular staining with dyes having general affinities for proteins or lipids. Overlapping spectral characteristics also have applications, for instance, emission by one fluorophore may be used to excite a second fluorophore through energy transfer when distances are sufficiently close.
Dyes that have been most widely used as markers for proteins and nucleic acid labeling include members of the xanthene, coumarin, cyanine and asymmetric cyanine dye families.
Xanthene dyes such as fluorescein are among the earliest dyes used for biological staining, where fluorescein was used to work out many of the techniques for labeling proteins and nucleic acids. The basic structure of fluorescein molecules is
Related xanthene compounds that have also been used as labels include rhodols and rhodamines. Their basic structure is
respectively. The R group attached to the central structure is typically a substituted phenyl group although, as described in U.S. Patent Publication 2003/0225247, aphenylic versions are also suitable as dyes.
Another family of dyes that have widespread use are coumarin derivatives. The basic structure of coumarin is
Typically, coumarin derivatives are dyes when R is an OH or an amine group. R may also be further modified such that enzymatic cleavage converts the R group into an OH or amine group. Such a proto-dye or dye precursor can be used as a marker for the presence of the enzyme that is capable of making the conversion. See, e.g., U.S. Pat. Nos. 5,696,157 and 5,830,912.
A large number of useful dyes are cyanines. The basic structure of cyanine dyes is
Major factors influencing the spectral qualities of these dyes is the number n, the nature of X and Y, and functional groups that extend the aromaticity of the dyes.
Other compounds that are functionally considered to be cyanine-type dyes are the merocyanine and styryl dyes, whose structures are:
respectively. See, e.g., U.S. Pat. No. 5,268,486.
There are a variety of atoms that have been used in the X and Y positions in the above cyanine dyes. These include carbon, sulfur, oxygen, nitrogen and selenium. When X or Y is a carbon, this portion of the dye is an indolinium moiety. When X or Y is substituted by sulfur, oxygen or nitrogen this portion is known as a benzothiazolium, benzoxazolium or a benzimidazolium moiety, respectively.
Another version of styryl dyes can have picoline or quinoline moieties instead of the benzazolium group, thereby having the structures:
respectively.
Asymmetric cyanine dyes contain one portion that is essentially the benzazolium portion of the cyanine dye family with a different aromatic compound connected to the benzazolium moiety by the methine bridge. Their structure is:
The aromatic moiety is generally a six membered aromatic or heteroaromatic ring.
Improvements to the various dyes described above have been made by substituting various groups onto the basic structure, e.g., on a carbon or nitrogen of the preceding structures or where H or R groups are featured. Additionally, other rings may be fused to various parts of the rings in the structures above, thereby generating more complex structures. These modifications have led to shifts in the excitation and emission characteristics of the dyes, such that there are now a large number of dyes with same basic structure but with different spectral characteristics. As described above, the cyanine dyes can have a general structure comprising two benzazolium-based rings connected by a series of conjugated double bonds. The dyes are classified by the number (n) of central double bonds connecting the two ring structures; monocarbocyanine or trimethinecarbocyanine when n=1; dicarbocyanine or pentamethinecarbocyanine when n=2; and tricarbocyanine or heptamethinecarbocyanine when n=3. The spectral characteristics of the cyanine dyes follow specific empirical rules. For example, each additional conjugated double bond between the rings usually raises the absorption and emission maximum about 100 nm. Thus, when a compound with n=1 has a maximum absorption of approximately 550 nm, equivalent compounds with n=2 and n=3 can have maximum absorptions of 650 nm and 750 nm respectively. Addition of aromatic groups to the sides of the molecules has lesser effects and may shift the absorption by 15 nm to a longer wavelength. Groups comprising an indolenine ring can also contribute to the absorption and emission characteristics. Using the values obtained with gem-dimethyl group as a reference point, oxygen substituted in the ring for the gem-dimethyl group can decrease the absorption and emission maxima by approximately 50 nm. In contrast, substitution of sulfur can increase the absorption and emission maxima by about 25 nm. R groups on the aromatic rings such as alkyl, alkyl-sulfonate and alkyl-carboxylate usually have little effect on the absorption and emission maxima of the cyanine dyes (see, e.g., U.S. Pat. No. 6,110,630).
As discussed above, alteration of spectral qualities is only one useful modification that can be made to a dye. In another aspect, modification of a dye by addition of a sulfonate group may increase the stability of many dyes and thereby resist “bleaching” after illumination. Additionally, modification of cyanine dyes by sulfonation decreases aggregation of target molecules labeled with those dyes (see, e.g., U.S. Pat. No. 5,569,766). Such modifications were also applied to xanthenes, coumarins and the non-benzazolium portion of asymmetric cyanine dyes (see, e.g., U.S. Pat. Nos. 5,436,134, 6,130,101 and 5,696,157). Modifications of dyes haves also been made to increase their affinity or selectivity towards binding to nucleic acids (see, e.g., European Patent Publication EP 0 231 495, U.S. Patent Publication 2003/0225247 and U.S. Pat. No. 5,658,751).
The utility of many of these dyes has been enhanced by synthesis of compounds with a reactive group that allows attachment of the dye to a target molecule. For instance, although cyanine dyes in themselves had been known for many years, it was only when derivatives were described with reactive groups (see, e.g., U.S. Pat. No. 5,268,486) that they found widespread use in labeling proteins and nucleic acids. Their versatility was then increased further by disclosure of other groups that could be used to attach cyanine dyes to suitable partners (see, e.g., U.S. Pat. No. 6,114,350 and U.S. Patent Publication 2003/0225247). An exemplary list of electrophilic groups and corresponding nucleophilic groups that can be used for these purposes are given in Table 1 of U.S. Pat. No. 6,348,596.
A variety of linker arms may be used to attach dyes to targets. Commonly used constituents for linkers are chains that contain varying amounts of carbon, nitrogen, oxygen and sulfur. Examples of linkers using some of these combinations are given in U.S. Pat. No. 4,707,440. Bonds joining together the constituents can be simple carbon-carbon bonds or they may be acyl bonds (see, e.g., U.S. Pat. No. 5,047,519), sulfonamide moieties (see, e.g., U.S. Pat. No. 6,448,008) and polar groups (see, e.g., U.S. Patent Publication 2003/0225247).
Because fluorescent dyes are used widely, e.g., for labeling nucleic acids, proteins and other molecules, there is an ongoing need for new dyes to provide more options for labeling methods and linker arm selections, spectral profiles and energy transfer (FRET) pair selection. The present invention addresses that need.